January 1, 2018;
The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.
Opacification of the ocular lens
, termed cataract, is a common cause of blindness. To become transparent, lens
fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1
post-transcriptionally controls key genes to regulate lens
fiber cell differentiation. Celf1
-targeted knockout mice and celf1
-knockdown zebrafish and Xenopus morphants have severe eye
spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1
by interacting with its 5'' UTR and mediating translation inhibition. Celf1
deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1
directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1
-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1
controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2
mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1
-regulated molecular mechanisms in lens
development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.
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Fig 1. Celf1 is required for vertebrate lens development.(A) In zebrafish, celf1 transcripts are detected in the lens at 1 day post fertilization (1dpf) by in situ hybridization (ISH). (B) In X. laevis, ISH indicates strong celf1 expression (arrow) in the embryonic St. 30 eye (arrow) and lens (indicated by broken line area). (C) In mouse, ISH shows strong Celf1 transcript expression in the lens at embryonic day 12.5. (D) In mouse lens, Celf1 protein is expressed at (D) E11.5 (E) E14.5 and (F) E16.5 predominantly in fiber cells (f) and to a lower extent in epithelial cells (e). (G, G’) In zebrafish, while control eyes are normal, celf1 knockdown (KD) results in microphthalmia and clouding of lens (asterisk) by 4dpf. (H, H’) In X. laevis, compared to control, celf1 KD results in microphthalmia. (I, I’) In mouse, compared to control, Celf1cKO/cKO lens exhibits severe cataract (asterisk). (J-K’) Compared to control, refraction errors (asterisk) are observed in Celf1cKO/cKO lens under dark-field and light-field microscopy. (L, L’) At E16.5 stage, the mouse Celf1cKO/cKO lens exhibits abnormal spaces (asterisk) in the fiber cell region. Scale bar in F is 75 μm.
Fig 2. Celf1cKO/lacZKI mouse exhibits mis-expression of key lens genes.(A) Microarray heat-maps representing genes mis-regulated in Celf1cKO/lacZKI lenses compared to control (left column, ±2.5 fold-change, p<0.05, total 34 genes, indicated by heatmap color gradients (left columns: green, down in Celf1cKO/lacZKI; red, up in Celf1cKO/lacZKI) and their respective enrichment in normal lens compared to whole-embryonic tissue as per iSyTE (right columns, lens-enrichment in fold-change indicated by red color intensity). (B) Differentially expressed genes (DEGs) in Celf1cKO/lacZKI lenses are plotted on the X-axis as down-regulated (circles) and up-regulated genes (triangles). On the Y-axis, DEGs are separated based on their lens-enrichment. Red and green color gradients represent high and low lens-enrichment, respectively. Genes down-regulated in Celf1cKO/lacZKI lenses are predominantly highly-lens enriched, while those up-regulated do not exhibit this trend.
Fig 3. Celf1 deficiency in mouse and fish causes fiber cell nuclear degradation defects.(A, B) Histological analysis of control and Celf1lacZKI/lacZKI mouse lenses at post natal day 4 (P4) stage shows abnormal presence of nuclei in centrally located fiber cells only in Celf1lacZKI/lacZKI mice. (A’, B’) High-magnification of the dotted-line area in A, B. Asterisk denote abnormally retained nuclei. (C, D) In zebrafish, compared to control, celf1KD lens exhibit abnormal presence of nuclei in the central fiber cell region. (C’, D’) High-magnification of the dotted-line area in E, F. Asterisk denote abnormally retained nuclei. (E) RT-qPCR analysis confirms significant Dnase2b down-regulation in Celf1cKO/lacZKI lenses compared to control. (F) RNA immunoprecipitation (RIP) and (G) cross-linked RNA immunoprecipitation (CLIP) shows Dnase2b to be enriched in Celf1-pulldown in wild-type mouse lens. (H) Celf1 over-expression in NIH3T3 cells, which carry Dnase2b 3’UTR downstream of a luciferase reporter, results in significant increase of luciferase mRNA. Abbr.: f.c., fold-change; NS, not significant. Asterisks in E, G, H indicate a p-value < 0.005.
Fig 4. Celf1-mediated post-transcriptional control of mitotic machinery components facilitates lens fiber cell nuclear degradation.(A, A’) Compared to control lens, phosphorylation of Lamin A/C (pLamin A/C) is reduced in Celf1cKO/lacZKI lens. Dotted-line areas of the fiber cell regions in control (B-D) and Celf1cKO/lacZKI mice (B’-D’) lenses are shown at high-magnification. Arrows in B, C indicate examples of high pLamin A/C expressing nuclei, while no nuclei are observed in D as this area represents a normal nuclear-free zone in the control lens. Asterisks in B’-D’ indicate reduced signals of pLamin A/C in Celf1cKO/lacZKI lens fiber cell nuclei. Note the presence of nuclei in D’ in the centrally located fiber cells due to the nuclear degradation defects in the Celf1cKO/lacZKI lens. (E, E’) Unlike in the control lens where p27Kip1 protein is restricted to cells of the transition zone and cortical fiber cells (arrow), high levels of p27Kip1 protein are detected in the entire fiber cell compartment (asterisks) including the central region in Celf1cKO/lacZKI lens. (F, F’) High-magnification of dotted-line areas in E and F. Asterisks indicate elevated signals of p27Kip1 protein. (G) Quantification of the p27Kip1 immunofluorescence signals from control and Celf1cKO/lacZKI lenses shows significantly increased p27Kip1 protein levels in Celf1cKO/lacZKI lenses. (H) Western blot analysis shows increased levels of p27Kip1 protein in Celf1cKO/lacZKI lens compared to control. (I) Compared to control, p27Kip1 mRNA levels are not significantly altered in Celf1cKO/lacZKI lens. (J, K) RIP and CLIP assays, respectively, identify p27Kip1 mRNA to be highly enriched in Celf1-pulldown in wild-type mouse lens. (L) A potential Celf1 binding GU-rich region in present in the mouse p27Kip1 5’ UTR. (M) Activity of firefly luciferase fused downstream of p27Kip1 5’ UTR is significantly elevated in Celf1-knockdown (Celf1-KD) mouse lens cell line compared to control (firefly luciferase normalized to Renilla luciferase (Rluc)). (N-O’) Compared to control, p21Cip1 mRNA and protein is abnormally elevated in Celf1cKO/lacZKI lens. Asterisk in O’ indicate high expression of p21Cip1 protein in the Celf1cKO/lacZKI lens. Abbr.: e, epithelial cells; f, fiber cells; tz, transition zone; NS, not significant. Scale bar in A’, E’, O’ is 75 μm and in D’ and F’ is 12 μm. Asterisks in G, K, M and N indicate a p-value < 0.0005, 0.005, 0.05 and 0.005, respectively.
Fig 5. Celf1 deficiency in mouse and fish causes defects in fiber cell morphology.(A) RT-qPCR analysis confirms significant Actn2 down-regulation in Celf1cKO/lacZKI lenses compared to control. (B) RNA immunoprecipitation (RIP) identifies Actn2 as an enriched transcript in Celf1-pulldown in P15 wild-type mouse lens. (C) Cross-linked RNA immunoprecipitation (CLIP) shows Sptb transcripts to be enriched in Celf1-pulldown in wild-type mouse lens. (D) RT-qPCR analysis shows that the high-abundant Sptb isoform (isoform 1 (ENSMUST00000021458)) is reduced, while the low-abundant Sptb isoform (isoform 2 (ENSMUST00000166101)) is abnormally elevated in Celf1cKO/lacZKI lenses. (E, E’) In mouse, phalloidin staining of lens tissue sections (stage P0) shows uniform F-actin deposition along the fiber cell margins in control, while Celf1cKO/lacZKI lenses exhibit abnormal pattern of F-actin (asterisk). (F, F’) In zebrafish, while control lens exhibits normal F-actin deposition, celf1KD lens (stage 4dpf) exhibits abnormal F-actin pattern (asterisk) in fiber cells. (G-H’) In mouse, scanning electron microscopy analysis of cortical and nuclear fiber cells shows disrupted cell organization (asterisk) in Celf1cKO/lacZKI lenses (stage P15). Scale bar in D’ is 75 μm and G’ is 2.5μM.
Fig 6. Model for Celf1-mediated post-transcriptional gene expression control in the lens.In normal lens development, Celf1 is required for nuclear degradation and proper cell morphology in fiber cell differentiation. Celf1 positively regulates the nuclease Dnase2b (being necessary for its high mRNA levels) and negatively regulates the cyclin-dependent kinase inhibitors p21Cip1 (being necessary for its low mRNA levels) and p27Kip1 (by inhibiting its translation into protein). Inhibition of p21Cip1 and p27Kip1 allows the activation of Cdk1, which phosphorylates Lamin A/C to initiate nuclear envelope breakdown in fiber cells. Thus, Celf1 controls the nuclease (Dnase2b) as well as its access to nuclear DNA, to regulate nuclear degradation in lens fiber cells. These findings show how mitotic machinery components–normally involved in nuclear envelope disassembly during cell division–are post-transcriptionally rewired by RNA-binding proteins to regulate cell differentiation in lens development. Additionally, Celf1 controls the splice isoform abundance of the membrane-organization protein Sptb (β-spectrin) and high transcript levels of the F-actin-binding protein Actn2 (α-actinin 2), to regulate fiber cell morphology. Abbr.: Epi, epithelium; TZ, transition zone; FC, fiber cells.